Pharmacological Disruption of the Notch Transcription Factor Complex

Pharmacological disruption of the Notch transcription factor complex

Rajwinder Lehala,b, Jelena Zarica, Michele Vigoloa,b, Charlotte Urechb, Viktoras Frismantasc,d, Nadine Zanggere, Linlin Caoa, Adeline Bergerf, Irene Chicoteg, Sylvain Loubéryh, Sung Hee Choii, Ute Kocha, Stephen C. Blacklowi, Hector G. Palmerg, Beat Bornhauserc,d, Marcos González-Gaitánh, Yvan Arsenijevicf, Vincent Zoetee,j, Jon C. Asterk, Jean-Pierre Bourquinc,d, and Freddy Radtkea,1

aSwiss Institute for Experimental Cancer Research, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland; bResearch & Development, Cellestia Biotech SA, 4057 Basel, Switzerland; cDepartment of Oncology, University Children’s Hospital Zürich, 8032 Zürich, Switzerland; dChildren’s Research Center, University Children’s Hospital Zürich, 8032 Zürich, Switzerland; eBioinformatics Core Facility, Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland; fUnit of Gene Therapy and Stem Cell Biology, Department of Ophtalmology, Jules-Gonin Eye Hospital, University of Lausanne, 1004 Lausanne, Switzerland; gStem Cells and Cancer Group, Vall d’Hebron Institute of Oncology, 08035 Barcelona, Spain; hDepartment of Biochemistry, Faculty of Sciences, University of Geneva, 1211 Geneva, Switzerland; iDepartment of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115; jComputer-Aided Molecular Engineering, Department of Oncology, Ludwig Institute for Cancer Research, University of Lausanne, CH-1005 Lausanne, Switzerland; and kDepartment of Pathology, Brigham and Women’s Hospital, and Harvard Medical School, Boston, MA 02115

Edited by Iva Greenwald, Columbia University, New York, NY, and approved June 1, 2020 (received for review December 23, 2019) Notch pathway signaling is implicated in several human cancers. sequential proteolytic cleavages of the receptor mediated by Aberrant activation and mutations of Notch signaling components metalloproteases of the ADAM family and the γ-secretase are linked to tumor initiation, maintenance, and resistance to multiprotein complex that liberate the Notch intracellular do- cancer therapy. Several strategies, such as monoclonal antibodies main (NICD). NICD subsequently traffics to the nucleus, binds against Notch ligands and receptors, as well as small-molecule the TF RBPJ, and recruits other coactivators, such as master- γ-secretase inhibitors (GSIs), have been developed to interfere mind proteins (MAML1-3) and p300, forming a transcription with Notch receptor activation at proximal points in the pathway. activation complex that initiates the expression of downstream However, the use of drug-like small molecules to target the down- target genes (2). stream mediators of Notch signaling, the Notch transcription acti- Next-generation sequencing of cancer genomes has identified APPLIED BIOLOGICAL SCIENCES vation complex, remains largely unexplored. Here, we report the numerous oncogenic gain-of-function mutations in NOTCH1 or discovery of an orally active small-molecule inhibitor (termed CB- NOTCH2 in B and T cell leukemias and lymphomas (3–5) and 103) of the Notch transcription activation complex. We show that solid cancers, such as adenoid cystic carcinoma (6) and breast CB-103 inhibits Notch signaling in primary human T cell acute lym- carcinoma (7, 8). These mutated or truncated genes encode phoblastic leukemia and other Notch-dependent human tumor cell proteins that are processed to NICD constitutively or have in- lines, and concomitantly induces cell cycle arrest and apoptosis, creased stability in their active forms, increasing the expression thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrange- Significance ments in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing The Notch signaling cascade is deregulated by oncogenic le- the dose-limiting intestinal toxicity associated with other Notch sions in human cancers and has therefore become an attractive inhibitors. Thus, we describe a pharmacological strategy that in- therapeutic target. Inhibitory monoclonal antibodies and small- γ terferes with Notch signaling by disrupting the Notch transcription molecule -secretase inhibitors have been developed to target complex and shows therapeutic potential for treating Notch- the pathway at the most proximal point of the cascade. Major driven cancers. hurdles to the therapeutic application of these Notch inhibitors have been prevalent dose-limiting toxicities in the intestine. Notch | small-molecule inhbitor | cancer Here we report identification and preclinical validation of a small molecule (CB-103) that inhibits the pathway at the level of the Notch transcription complex without causing intestinal ranscription factors (TFs) are key mediators of cellular toxicity. Its properties and mechanism of action provide CB-103 Tprocesses and cell states. In cancer, TFs are commonly with a more favorable therapeutic index than other types of deregulated indirectly by aberrant upstream signaling cascades Notch targeting agents, a feature that is currently being tested or directly by pathogenic mutations and or translocations. Al- in cancer patients. though in principle an attractive class of therapeutic targets, TFs are largely considered undruggable due to the absence of surface Author contributions: R.L., S.C.B., H.G.P., B.B., M.G.-G., Y.A., J.-P.B., and F.R. designed pockets amenable to effective targeting with small molecules. research; R.L., J.Z., M.V., C.U., V.F., L.C., A.B., I.C., S.L., S.H.C., U.K., H.G.P., and V.Z. per- Thus, most currently available targeted cancer therapeutics aim formed research; R.L., J.Z., M.V., C.U., V.F., N.Z., L.C., A.B., I.C., S.L., S.H.C., U.K., S.C.B., H.G.P., B.B., M.G.-G., Y.A., V.Z., J.C.A., J.-P.B., and F.R. analyzed data; and R.L., J.Z., N.Z., at inhibiting oncogenic signaling pathways at the most proximal U.K., S.C.B., M.G.-G., Y.A., V.Z., J.C.A., and F.R. wrote the paper. part of the cascade, either through antibodies against ligands or Competing interest statement: R.L. and F.R. are cofounders of Cellestia Biotech AG, and surface receptors, or using small molecules that inhibit the en- R.L., M.V., and C.U. are employees of the company. zymatic activities of receptor-associated kinases. This article is a PNAS Direct Submission. The Notch signaling cascade is one example of a pathway that Published under the PNAS license. has emerged as a rational therapeutic target in several cancers. Data deposition: The SLAM-seq data reported in this paper have been deposited in the In the adult, Notch signaling in progenitor and stem cells regu- Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession lates tissue homeostasis, self-renewal, and differentiation in no. GSE148228). several organs and cell types, including the intestine, vasculature, 1To whom correspondence may be addressed. Email: [email protected]. and hematopoietic system (1). This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Notch signaling is initiated through the interaction of a re- doi:10.1073/pnas.1922606117/-/DCSupplemental. ceptor and ligand on neighboring cells. This event results in

www.pnas.org/cgi/doi/10.1073/pnas.1922606117 PNAS Latest Articles | 1of10 Downloaded by guest on September 30, 2021 of target genes that deregulate cell growth and survival. Several A screen of 67,253 compounds from commercially available strategies, such as monoclonal antibodies (MAbs) against Notch libraries identified 341 compounds with Notch inhibitory activity. ligands (9–11) and receptors (12, 13), and small-molecule Computer-aided self-organizing mapping programs that cluster γ-secretase inhibitors (GSIs) (14, 15), have been developed to compounds based on structural similarities allowed reduction of block Notch receptor activation at proximal points in the path- the number of hits to 98, of which 33 were validated by a sec- way. Whereas MAbs have the advantage of specifically inhibiting ondary screen in the same coculture assay. These compounds individual ligands or receptors, GSIs are pan-Notch inhibitors were then assayed for their ability to block signaling driven by a that block signaling through all four Notch receptors (16). GSIs dominant active form of the Notch1 receptor (N1-ICD) were originally developed for treating Alzheimer disease because (Fig. 1C). Importantly, this approach enables identification of they inhibit the cleavage of amyloid precursor protein. In addi- compounds that act downstream of the γ-secretase–mediated S3 tion, they also block the proteolytic cleavage step (S3 cleavage) cleavage event. Employing this strategy, we identified the com- that generates NICD, leading to their widespread experimental pound CB-103 (Fig. 1D). To assess whether CB-103 specifically use as Notch inhibitors (1, 14). However, GSIs also block the inhibits Notch1 or is also active against other Notch receptors, processing of more than 90 other substrates, which may com- we tested the ability of CB-103 to inhibit Notch2-, Notch3-, and plicate the interpretation of results produced by GSIs (16). Al- Notch4-mediated signaling using the in vitro coculture assay. though both MAbs and GSIs have shown beneficial effects in CB-103 inhibited Notch signaling mediated by each of the re- preclinical Notch-driven tumor models and clinical studies (12, ceptors tested in a dose-dependent manner (Fig. 1 E and F). As 17–21), none of these Notch inhibitors have been clinically ap- was observed for N1-ICD (Fig. 1G), CB-103 was also able to proved, largely due to on-target dose-limiting toxicities of the block the activity of the dominant active forms of Notch2, intestinal epithelium (22, 23). Treatment of patients with GSIs is Notch3, and Notch4 (N2-ICD, N3-ICD, and N4-ICD, re- frequently associated with diarrhea, vomiting, and nausea, which spectively) (Fig. 1H). Overall, CB-103 inhibited both ligand- may be severe (24, 25). To avoid this toxicity, clinical trials in dependent and ligand-independent Notch activation in cell- Notch-driven cancers have relied on intermitting dosing of GSIs based assays, with IC50 values ranging from 0.9 to 3.9 μM (14). However, the question remains as to whether intermittent (Fig. 1 F and H); CB-103 did not inhibit Wnt or Hedgehog sig- dosing strategies sustain Notch inhibition long enough to achieve naling using reporter assays (SI Appendix, Fig. S1). therapeutic efficacy. In light of the above data, we hypothesized that CB-103 acts to There have also been attempts to target the pathway down- prevent Notch-mediated transcription and thus sought to sub- stream of the γ-secretase–mediated activation of Notch recep- stantiate this prediction. Experiments using a N1-ICD–GFP fu- tors. One is based on the finding that truncated forms of sion protein indicated that CB-103 does not prevent nuclear MAML1 that bind the RBPJ–NICD complex but lack the ability translocation of N1-ICD (Fig. 1I), excluding impaired trafficking to recruit other coactivators function in a dominant-negative as a mechanism of action. An alternative possibility is that it manner (26–28). Based on this concept, Bradner and col- interferes with a functional assembly of the transcription com- leagues (29) synthesized a stapled peptide named SAHM1 plex. Consistent with this, expression of increasing amounts of (stapled α-helical peptide derived from MAML1) designed to the cofactor MAML1 counteracted the inhibitory effect of CB- mimic dominant-negative forms of MAML1. However, de- 103 (Fig. 1J), suggesting that CB-103 impairs the recruitment and veloping drug-like stapled peptides as therapeutics remains assembly of components of the Notch transcription complex. challenging due to manufacturing, stability, and pharmacokinetic issues. Another approach utilized screens to identify the small CB-103 Inhibits Growth of Notch-Addicted Human T Cell Acute molecule Mastermind recruitment-1 (IMR-1), which is also Lymphoblastic Leukemia Cell Lines through Modulation of the proposed to have dominant-negative MAML-like activity (30). Notch Transcription Complex. To further establish CB-103 as a Finally, a recent report describes the identification of a small bona fide Notch inhibitor, we tested its ability to directly inhibit molecule that blocks the interaction between RBPJ and SHARP, the growth of Notch-dependent cancer cells by initially focusing a protein that forms a corepressor complex with RBPJ (31). on T cell acute lymphoblastic leukemia (T-ALL). More than However, this approach does not inhibit NOTCH signaling, but 50% of T-ALL patients harbor activating NOTCH1 mutations rather leads to derepression of NOTCH target genes (31). Al- resulting in increased Notch signaling (3). Treatment of the though all of these Notch TF complex-modulating compounds NOTCH1-mutated human T-ALL cell line RPMI-8402 and the show inhibitory activities in cellular assays, it remains to be de- NOTCH3-driven human TALL-1 cell line with CB-103 or a GSI termined whether these inhibitors possess drug-like properties, resulted in down-regulation of the Notch target genes HES1, as none of these compounds have been tested in clinical trials. MYC, and DTX1 (SI Appendix, Fig. S2 A and B), as well as down- Here, we report the discovery and preclinical validation of an regulation of NOTCH1 (SI Appendix, Fig. S2C). Interestingly, orally active small molecule [6-(4-(tert-butyl)phenoxy)pyridine-3- protein levels of N1-ICD were unaffected by short-term (6 h) amine, termed CB-103] that interferes with the function of the treatment with CB-103 (SI Appendix, Fig. S2C), but were sig- Notch transcription complex. CB-103 induces loss-of-function nificantly reduced at later time points (24 and 48 h) (SI Appen- phenotypes in flies and mice and has antitumor activity in xe- dix, Fig. S2 D and E). In contrast, protein levels of RBPJ were nograft models of Notch-addicted human leukemia and carci- unaffected (SI Appendix, Fig. S2E). Simultaneous treatment of noma without causing gut toxicity. CB-103 possesses excellent RPMI-8402 cells with CB-103 and a proteasome inhibitor did drug-like properties and is currently being evaluated in a phase not restore N1-ICD levels, suggesting that CB-103 does not I clinical trial in cancer patients. impact N1-ICD protein levels by enhancing proteasomal degra- dation (SI Appendix, Fig. S2F). As NOTCH1 exhibits positive Results autoregulation in T-ALL cells, these results are consistent with a Identification of CB-103 as a Notch Inhibitor. Seeking to discover fall in N1-ICD levels due to inhibition of NOTCH1 transcription. small-molecule inhibitors of Notch signaling, we developed a In addition, CB-103 induced profound cell growth inhibition in cell-based coculture assay amenable to high-throughput screen- both RPMI-8402 and T-ALL1 cells (SI Appendix,Fig.S2G and H). ing of chemical compound libraries. This screen utilized a co- In contrast, growth of the Notch-independent HeLa cell line was culture system consisting of “signal-receiving” HeLa cells unaffected by either CB-103 or GSI treatment (SI Appendix,Fig. expressing Notch1 in combination with a Notch-responsive lu- S2I). Global gene-expression analysis of CB-103-treated NOTCH1- ciferase reporter, and “signal-sending” HeLa cells expressing the mutated T-ALL cell lines KOPT-K1 and HBP-ALL further con- Notch ligand Delta-like 4 (DL4) (Fig. 1 A and B). firmed down-regulation of N1-ICD–driven growth-promoting

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Fig. 1. Identification of CB-103 as an inhibitor of the Notch transcription activation complex. (A) Schematic of the DL4 Notch1 (N1) coculture assay used high- throughput screening in HeLa cells. Ligand-receptor mediated pathway activation was measured using a Notch-responsive luciferase reporter. (B) Assay validation of the DL4-N1 coculture screening with indicated Z′ value. Bar graph shows result of one representative 384-well plate, half treated with 10 μMof the GSI DAPT and half with vehicle control. (C) Schematic representation of the N1-ICD–driven luciferase reporter assay used to counter screen-validated hits of the primary screen. (D) Chemical structure of CB-103. (E) Bar graphs show concentration-dependent assessment of CB-103 and GSI (DAPT) to DL4- Notch1–mediated signaling in the RBPJ-driven reporter and coculture assay. Luciferase activity was measured 24 h after treatment. (F) Dose–response curves for mNotch1, mNotch2, mNotch3, and mNotch4 activation following treatment of cocultures with CB-103. (G) Bar graph shows concentration dependent assessment of CB-103 and GSI (DAPT) to inhibit N1-ICD–mediated, RBPJ-driven reporter assay. (H) Bar graphs show results of a second independent N1-ICD+ PDX model xenografted into NSG mice. Vehicle control and CB-103 treatment groups were subdivided into high (Upper) and low tumor burden groups (Lower) based on >20% and <2% of T-ALL cells detected in peripheral blood by flow cytometric analysis at treatment initiation. (I) Nuclear localization of N1-ICD–GFP fusion protein in HeLa cells in the presence of DMSO or CB-103 (representative of three independent experiments). (Magnification, 100×.) (J) N1-ICD–induced luciferase activity in the presence of CB-103 and increasing amounts of MAML1.

genes, including MYC, the main oncogenic driver directly regulated and then investigated a possible mechanism of insensitivity by Notch in T-ALL (32, 33) (SI Appendix,Fig.S3). (mutation) by exome sequencing, motivated by the hypothesis To gain insight into the molecular mechanism underlying CB- that drug insensitivity could occur through gene mutations af- 103 inhibition of Notch-mediated transcription, we generated fecting the drug’s targets (34). Exome sequencing of CB-103 RPMI-8402 T-ALL cell lines with reduced sensitivity to CB-103 insensitive RPMI-8402 T-ALL cells identified a G193R mutation

Lehal et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 30, 2021 within the BTD domain of RBPJ. Importantly, engineered ex- to RBPJ, providing an explanation for how CB-103 impairs the for- pression of a V5-tagged RBPJG193R mutant gene in parental RPMI- mation and activity of the NOTCH1 transcription complex. 8402 cells shifted the IC50 for CB-103 from 2.6 μMto>100 μM, In further support of this model, CB-103 also interfered with whereas expression of V5-tagged WT RBPJ had minimal effects, recovery of N1-ICD in immunoprecipitates prepared from indicating that this specific single amino acid change is sufficient to RPMI-8402 cells expressing V5-WT-RBPJ, but not from cells confer insensitivity to CB-103 treatment (Fig. 2). expressing V5-RBP-JG193R protein (Fig. 3 A and B). Further- Next, we performed computational docking studies. CB-103 more, CB-103 reduced the occupancy of RBPJ and N1-ICD on was docked on the NOTCH1 transcription complex/HES1 pro- genomic Notch-response elements associated with the Notch target moter DNA system to determine a possible binding mode on the genes HES1, DTX1,andMYC in RPMI-8402 cells expressing native structure (35). Among the calculated binding modes, one V5-WT-RBPJ but not in cells expressing the V5-RBP-JG193R mutant confirmed the BTD domain of RBPJ as possible binding site for (Fig. 3C). Taken together, these results strongly suggest that CB-103 CB-103 and identified several key RBPJ amino acid residues inhibits Notch-mediated transcription by interfering with assembly (Fig. 2A). Expression of engineered forms of RBPJ-bearing of the Notch transcription complex. mutations in these residues—specifically V5-tagged RBPJF196A, The majority of Notch/RBPJ binding sites that mediate acute V5-tagged RBPJL245A, or V5-tagged RBPJL248A, but a control changes in gene expression are found in enhancers (36). These mutant V5-tagged RBPJG194R—in parental RPMI-8402 cells genomic response elements are of two distinct types, one con- conferred resistance to CB-103 (Fig. 2 B and C and SI Appendix, taining monomeric RBPJ sites and the second dimeric Fig. S4). Thus, combined docking and mutational analysis sup- head-to-head RBPJ sites separated by 15 to 17 base pairs, an port the binding of CB-103 to this pocket in the BTD domain. Notably, element called a sequence-pair site (SPS) that supports loading this pocket is also important for binding of the N1-ICD RAM domain of dimeric Notch TF complexes. SPS-mediated Notch signaling

Fig. 2. Single amino acid mutations within the BTD domain of RBPJ cause unresponsiveness to CB-103 in RPMI-8402 cells. (A) Experimental structure of the NOTCH1 transcription complex on the HES1 promoter DNA sequence (PDB ID code 3V79) (64). RBPJ (orange), domains are show in ribbon representation. HES1 backbone is shown in ribbon representation, with nucleotides displayed as tubes. Green arrows indicate predicted amino acids important for binding of CB-103 within the BTD domain of RBPJ. (B) Graph shows dose–response curves of CB-103–treated parental, RPBJwt–V5- and RBPJG193R–V5-, RBPJL245A–V5-, RBPJL248A–V5-, RBPJF196A–V5-, and G194R RBPJ –V5-expressing RPMI-8402 cells. Cells were treated with CB-103 for 3 d. IC50 values for respective cell lines are indicated. (C) Bar graphs show percentage of apoptotic cells of parental, RBPJwt–V5- and RBPJG193R–V5-, RBPJL245A–V5-, RBPJL248A–V5-, RBPJF196A–V5-, and RBPJG194R–V5-expressing RPMI-8402 cells treated with DMSO or CB-103 (10 μM) for 72 h. Statistical analysis was performed using two-tailed t test (***P < 0.0005, **P < 0.007; ns, not significant).

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1922606117 Lehal et al. Downloaded by guest on September 30, 2021 RPMI-8402 RPMI-8402 A input IP: V5 B input IP: V5 G193R G193R RBPJ WT -V5 RBPJ WT -V5 RBPJ -V5 RBPJ -V5 CB-103 CB-103 N1-ICD N1-ICD

RBPJ - V5 RBPJ - V5 MYC MYC TUBULIN TUBULIN

RPMI-8402 G193R C RBPJ WT -V5 RBPJ -V5 0.2 0.1 0.1 0.1 DMSO ns 0.3 ns 0.1 **** ** CB-103 HES1 input %

0.0 0.0 0.0 0.0 0.0 0.0

0.06 **** 0.04 **** 0.1 0.06 ns 0.1 ns 0.06 DTX1 input %

0.00 0.00 0.0 0.00 0.0 0.00

0.05 0.025 ****0.025 ** 0.025 0.05 ns 0.04 ns MYC input %

0.000 0.00 0.0 0.00 0.0 0.0 antibody: N1-ICD 5V GgI N1-ICD 5V GgI

Fig. 3. CB-103 inhibits assembly of the RBPJ–NICD transcription complex. (A and B) RPMI-8402 cells expressing either RBPJwt-V5 (A) or RBPJG193R-V5 mutant protein (B) were treated with vehicle control (−) or CB-103 (+), 10 μM for 14 h and subjected to immunoprecipitation using a V5-specific antibody. N1-ICD wt G193R immunoprecipitates were assessed by Western blot. Western blot analysis for MYC expression was performed on input: Parental, RBPJ -V5, or RBPJ -V5 APPLIED BIOLOGICAL SCIENCES mutant protein expressing RPMI-8402 cells. (C) DMSO- or CB-103–treated RPMI-8402 cells expressing either RBPJwt-V5 or RBPJG193R-V5 protein were subjected to ChIP. RBPJ binding regions from NOTCH target genes HES1, DTX1,andMYC were PCR-amplified from input and precipitated DNA. Location of the PCR amplicons is schematically illustrated to the left (red dash). Results are expressed as percentage relative to input. Shown are mean ± SD (n ≥ 3). Statistical analysis was performed using one-way ANOVA (****P < 0.0001, **P < 0.009; ns, not significant).

is important for T cell maturation and leukemic transformation S7). In the same assays, we failed to identify any effect of IMR-1 on but dispensable for T cell fate specification in mice (37, 38). To MYC levels or RPMI-8402 cell growth at doses up to 10 μM(SI determine whether CB-103 preferentially inhibited elements Appendix,Fig.S7). Thus, CB-103 acts by a markedly different containing SPSs versus head-to-tail–oriented RBPJ binding sites, mechanism than SAHM1 and IMR-1 with respect to the expected we performed luciferase reporter gene assays. Although SPS- activities of direct Notch transcription complex. driven reporters elicited a much stronger signal compared to head-to-tail–oriented RBPJ-driven promoters, both were equally CB-103 Function In Vivo Recapitulates Genetic Notch Loss-of-Function sensitive to CB-103. Thus, CB-103 inhibits both monomeric and Phenotypes without Causing Gut Toxicity. Prior to investigating the dimeric Notch1 TF complexes, which agrees with the proposed in vivo activity of CB-103, we profiled the compound to de- mode of action of CB-103 (SI Appendix, Fig. S5). termine its drug-like and ADME/PK (absorption, distribution, Next, we performed kinetic gene-expression analysis using metabolism, excretion, and pharmacokinetic) properties (SI SLAM-seq [thio(SH)-linked alkylation for the metabolic se- Appendix, Fig. S8). Then we assessed CB-103’s ability to affect a quencing of RNA] on vehicle and CB-103–treated RPMI-8402 variety of Notch-dependent cellular processes. First, we studied T-ALL cells to investigate potential differences in the CB-103 the effect of CB-103 on the development of mechanosensory sensitivity of promoter- and enhancer-driven Notch target genes. organs in Drosophila, which consist of four distinct lineages Pathway analysis from the Hallmark collection revealed E2F (socket, shaft, sheath, and neuron) derived from a single sensory targets, MYC targets, and PI3K AKT MTOR signaling as being organ precursor cell (39). Notch signaling loss induces aberrant the most rapidly down-regulated pathways. Interestingly, the Notch sensory organ lineages, including lineages composed of two target genes DTX1, HES1, NOTCH3,andNOTCH1,whichare neurons and no sheath cells, indicative of sheath-to-neuron regulated by response elements found in promoters or intragenic transformation (39). Treatment of developing sensory organs enhancers, were down-regulated faster than target genes that are with CB-103 resulted in increased numbers of neurons, similar to regulated by long-distance enhancers—such as MYC, GIMAP1,-5, the effect produced by GSI (DAPT), demonstrating that CB-103 -6,and-8—suggesting that different Notch target genes may have inhibits Notch-dependent cell fate specification in flies (SI Ap- varying sensitivities and or kinetics of response to CB-103 (SI Ap- pendix, Fig. S9). pendix,Fig.S6). We extended these findings to mammals and investigated how In previous studies, the stapled peptide and small molecules CB-103 affects five Notch-dependent phenotypes in mice: 1) SAHM1 and IMR-1 were also claimed to target the Notch tran- Development of splenic marginal zone B (MZB) cells, 2) thymic scription complex (29, 30); we therefore compared their activities T cell development, 3) generation of Esam+ dendritic cells, 4) with CB-103 in reporter gene assays and in Notch-driven T-ALL sprouting of endothelial cells, and 5) induction of goblet cell cells. The activity of SAHM1 in reporter gene assays was in- differentiation in the small intestine. Each of these processes distinguishable from unstapled control peptide; and although strictly require Notch signaling (40–46). As expected, treatment SAHM1 was cytotoxic, unlike CB-103, it did not down-regulate with CB-103 (SI Appendix, Fig. S10A) resulted in a dramatic, MYC expression in RPMI-8402 T-ALL cells (SI Appendix,Fig. reversible reduction of B220+CD21hiCD23int MZB cells (SI

Lehal et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 30, 2021 Appendix, Fig. S10B), mimicking phenotypes caused by loss of treatment with either LY3039478 or CB-103, whereas only CB-103 Notch2 (38) or Dll1 (39). Osmotic pump-mediated delivery to elicited growth arrest in DND-41 cells expressing N1-ICD (SI Ap- maintain sustained exposure to CB-103 resulted in a concentration- pendix,Fig.S14A). CB-103 sensitivity was in turn rescued by ectopic dependent reduction of MZB cells and revealed that a level of expression of MYC (SI Appendix,Fig.S14B), consistent with prior 700 ng/mL (2.8 μM) in plasma is sufficient to inhibit Notch signaling work implicating MYC as a key Notch target gene in T-ALL cells (51). (SI Appendix,Fig.S10C and D). Similarly, we observed impaired Next, we investigated the ability of CB-103 to inhibit growth of thymic T cell development (SI Appendix,Fig.S11A), reduced Notch-dependent cancers that are resistant to MAbs and GSI − numbers of Esam+CD11c+CD8 dendritic cells (SI Appendix,Fig. therapy. We focused on triple negative breast cancer (TNBC), S11B), and increased endothelial cell sprouting in CB-103–treated since ∼10% have rearrangements in NOTCH1 and/or NOTCH2 WT mice (SI Appendix,Fig.S12). that lead to constitutive, ligand-independent Notch activation (7, In notable contrast, CB-103 treatment did not produce the 8). As predicted, growth of the GSI-resistant HCC1187 cell line expected intestinal phenotype. Genetic deletion of genes carrying a NOTCH2 rearrangement (7) was inhibited by CB-103 encoding Notch1 and Notch2 receptors or Dll1 and Dll4 ligands treatment but not by RO4929097, a GSI previously assessed in or Rbpj, as well as sustained treatment with potent GSIs, result in clinical phase I/II studies (8) (Fig. 5A). We subsequently estab- goblet cell metaplasia and reduced proliferation of crypt pro- lished a stable luciferase reporter line to determine the ability of genitor cells (40, 41, 47). Goblet cell metaplasia-associated in- CB-103 to inhibit growth of HCC1187 following xenotransplantation. testinal toxicity is one of the main dose-limiting “adverse events” CB-103–treated animals showed remarkable growth inhibition and in clinical trials using Notch-targeting agents, such as GSI or reduced tumor burden compared to vehicle-treated animals (Fig. 5B). antagonistic Notch receptor antibodies (1). We treated mice with CCDN1, a known Notch target in breast cancer (52, 53), was down- vehicle control, CB-103, or the GSI LY3039478. Mice treated regulated in tumors from CB-103–treated animals compared to con- with vehicle control or CB-103 were analyzed after 1 wk and 4 wk trols (Fig. 5C). Moreover, CB-103–mediated tumor growth inhibition of treatment, while mice treated with LY3039478 were analyzed was accompanied by reduced Ki67 and increased Caspase after 1 wk due to treatment-related morbidity. Alcian blue and 3 staining, while CD31 staining was similar in vehicle- and CB- – Ki67 staining revealed a dramatic increase in goblet cell numbers 103 treated animals (SI Appendix, Fig. S15). Our data provide and a highly significant reduction in crypt cell proliferation in proof-of-concept that CB-103 can inhibit growth of “Notch- LY3039478-treated animals. In contrast, CB-103–treated ani- addicted” cancer cells expressing mutated forms of Notch that mals showed only a moderate increase in goblet cell numbers cannot be targeted with agents that act upstream of the Notch + and a moderate reduction in Ki67 crypt cells (Fig. 4). TF complex. Furthermore, patient-derived xenotransplanation To investigate the basis of the milder gut phenotype in CB- (PDX) experiments using a NOTCH1+ oxaliplatin-resistant co- 103–treated mice, we studied its effects on key downstream lorectal cancer sample (54) revealed that CB-103 resensitizes this target genes. In the small intestine, Notch signaling directly tumor to oxaliplatin treatment in vivo (Fig. 5D). regulates the expression of the stem cell marker gene Olmf4 and To extend these findings to primary human cancers, we in- members of the Hes family of TFs, which repress the transcrip- vestigated the ability of CB-103 to inhibit the growth of 19 pri- tional master regulator for secretory cells, Atoh1 (48–50). In situ mary T-ALLs, the human tumor with the highest frequency of hybridization studies revealed that the expression of Olmf4 and Notch gain-of-function mutations, in a coculture model (47). Hes1 was nearly completely abrogated by CB-103 and LY3039478, Dose–response profiles indicated that CB-103 induced growth thereby demonstrating Notch inhibitory activity of these agents in inhibition in ∼50% of the cases tested with IC50 values in the the gut. In contrast, LY3039478-treated animals had significantly submicromolar range (Fig. 5E). Importantly, the ability of CB- higher levels of Atoh1 transcripts compared to vehicle- and CB- 103 to reduce tumor growth correlated strictly with Notch acti- 103–treated animals, which showed only a small increase in Atoh1 vation status, as only tumors containing N1-ICD responded to transcripts relative to vehicle-treated animals (Fig. 4B). To exclude CB-103 treatment. Furthermore, growth inhibition induced by possible confounding influences of variation in absorption or CB-103 was associated with the presence of elevated levels of pharmacokinetic properties in vivo, we tested these compounds in N1-ICD pretreatment and decreased N1-ICD levels posttreat- intestinal organoid cultures created from WT mice. LY3039478-treated ment, but did not strictly correlate with the mutation status of organoids had a very different morphology than CB-103–treated cul- NOTCH1 or FBXW7 (Fig. 5F). These results indicate that CB- tures and showed increased goblet cell differentiation and decreased 103 selectively inhibits the growth of T-ALLs with ongoing proliferation compared to vehicle- or CB-103–treated organoids (SI NOTCH1 activation and supports prior studies suggesting that Appendix,Fig.S13A). We again observed strong inhibition of the Notch the level of N1-ICD predicts tumor response to Notch pathway pathway, as indicated by down-regulation of direct Notch target genes inhibitors (44). In line with these ex vivo results, CB-103 pro- Olmf4, Hes1, Hes3,andHes5 to a similar degree by both CB-103 and longed the survival of mice bearing a NOTCH1-mutated T-ALL LY3039478, whereas Atoh1 transcripts were up-regulated to higher PDX model compared to vehicle treatment (Fig. 5 G, Left), an levels in LY3039478-treated organoids (SI Appendix,Fig.S13B). These antitumor effect that was also associated with decreased N1-ICD results show that while both compounds block Notch signaling in the levels (Fig. 5 G, Right). A second independent N1-ICD+ T-ALL intestine in vivo and in intestinal organoids, goblet cell metaplasia and PDX model also showed significantly reduced tumor burden in profound growth inhibition were only observed with LY3039478 mice with either high or low tumor burden (>20% and <2% treatment, presumably due to the higher expression levels of Atoh1. leukemic blasts in the peripheral blood) at treatment initiation, as indicated by percentages of circulating huCD45+CD7+ CB-103 Blocks Tumor Growth of GSI-Resistant Cancers. A potential T-ALL blasts after CB103 treatment (Fig. 5H). advantage of a small-molecule inhibitor of the Notch TF complex is the ability to block Notch signaling in tumors that are Discussion resistant to Notch-directed MAbs or GSIs by virtue of Notch In the last decade unbiased next-generation sequencing efforts of gene rearrangements that lead to γ-secretase independent Notch human cancer specimens have identified activating genetic ab- activation. As a proof-of-principle, we studied via lentiviral ex- errations in NOTCH genes in a broad spectrum of cancers, in- pression of constitutively nuclear N1-ICD whether CB-103 could cluding T-ALL, adenoid cystic carcinoma, chronic lymphocytic inhibit growth of Notch-dependent human DND-41 T-ALL cells leukemia, MZB cell lymphoma, and breast cancer (3, 6, 7, 55). rendered resistant to agents (such as GSI) that act upstream of Moreover, many preclinical studies have implicated Notch signaling the Notch TF complex. As expected, parental DND-41 cells in almost all hallmarks of cancer, highlighting why this signaling exhibited reduced growth inhibition and Notch signaling following pathway is an intriguing but complex therapeutic target (56).

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1922606117 Lehal et al. Downloaded by guest on September 30, 2021 A vehicle CB-103 GSI B 100 **** vehicle **** 80 CB-103 60 GSI 40 ****

in vili/field 20 Alcian blue

No. of pos.cells 0

150 **** **** **** 100

Ki67 50 crypt cells % of positive 0

150 **** ns **** 100

Olfm4 50 score 4

% of positive crypts 0

80 **** **** 60 ns 40 score 4

Hes1 20

% of pos.cells/ field 0

150 ****

**** 100 ****

Atoh1 50 % of pos.cells/ field 0

Fig. 4. CB-103 reduces Notch target gene expression in the small intestine without causing goblet cell metaplasia. (A) Alcian blue (Top row), Ki67 staining ( second row), and in situ hybridizations for expression of Notch target genes Olmf4, Hes1, and Atoh1 of representative sections from the proximal small intestine of vehicle, CB-103– (20 mg/kg 2× day) and GSI- (LY3039478, 20 mg/kg 2× day) treated mice are shown. Mice were treated with CB-103 up to 4 wk and analyzed at either 1 or 4 wk postadministration. Analysis at two time points revealed comparable results. CB-103 treatment at 4 wk and GSI treatment at 1wk is depicted. (B) Bar graphs show quantification of indicated stainings and in situ hybridizations. The number of Alcian blue-positive cells per crypt-villus unit APPLIED BIOLOGICAL SCIENCES + and Ki67 cells per crypt is expressed as percentage of positive cells per field, of vehicle (n = 2 mice, 130 crypt-villus units for Alcian blue and 462 crypts for Ki67), CB-103 (n = 6 mice, 375 crypt-villus units and 430 crypts for Ki67), and GSI (n = 3 mice, 135 crypt villus-units and 330 crypts for Ki67) -treated animals. In situ hybridization of Olmf4, Hes1, and Atoh1 expression was quantified and is expressed as percentage of positive crypts for Olmf4 (score 4, n = 2 mice for vehicle-treated animals, 190 crypts; n = 6 mice for CB-103 treatment, 600 crypts; n = 3 mice for GSI-treated animals, 300 crypts), percentage of positive cells per crypt for Hes1(score 4, n = 2 mice for vehicle-treated animals ,140 crypts; n = 6 mice for CB-103 treatment, 170 crypts; n = 3 mice for GSI-treated animals, 110 crypts) and percentage of positive cells per field for Atoh1 expression (n = 2 mice, vehicle group, 150 crypt-villus units; n = 6 mice, CB-103 group, 340 crypt- villus units; n = 3 mice, LY3039478 group, 220 crypt-villus units). (Scale bars for Alcian blue, 50 μm; for other slides, 20 μm.) Statistical analysis was performed using unpaired t test (****P < 0.0001; ns, not significant).

Herein we describe a cell-based high-throughput screen that target genes, such as HES1, DTX1, and MYC (Fig. 3). In addi- led to the identification and characterization of a small-molecule tion, computational docking studies identified the BTD domain inhibitor (CB-103) of the Notch cascade. CB-103 is a pan Notch of RBPJ as potential binding pocket for CB-103, and accurately inhibitor as it can block Notch-mediated signaling of all four predicted amino acids residues in RBPJ that when mutated Notch receptors (Fig. 1), similar to GSIs. Commonly used GSIs confer resistance to CB-103. Taken together, the computational block Notch signaling by inhibiting the proteolytic activity of the docking studies, mutational analysis, as well as pulldown and γ-secretase multiprotein complex, which cleave Notch receptors ChIP experiments are in agreement with CB-103 acting as a at the S3 site within the transmembrane domain. In contrast to direct inhibitor of the Notch transcription complex. GSIs, CB-103 evidently inhibits the pathway at the most down- Oncogenic Notch signaling can be triggered by a variety of stream level—the Notch transcriptional complex—based on its mutations, including single nucleotide substitutions, small in- ability to block GSI-insensitive dominant active forms of NICD. sertion/deletion mutations, and rearrangements of Notch genes. Further evidence comes from the generation of a T-ALL cell line One advantage of CB-103 over currently available Notch inhib- that is resistant to CB-103. The rationale of this experimental itors is that it is active against tumor cells bearing any of these design was that drug insensitivity can be afforded through mu- types of mutations, which is not uniformly true of other Notch tations that circumvent the inhibitory activity by the drug, mu- inhibitors. Mutated Notch receptors found in tumors with Notch tations that have the potential to reveal mechanistic insights both gene rearrangements lack ectodomains and therefore do not rely into the actions of the drug and its target (34). In the past this on ligand for activation and cannot be targeted with blocking approach has been used successfully to identify drug targets for antibodies. Furthermore, ∼5% of TNBCs and 52% of glomus BI 2536, a Polo-like kinase1 inhibitor (50), as well as for the tumors have gene rearrangements in NOTCH2 (7, 8, 58) in which proteasome inhibitor Bortezomib, which is clinically used to treat N2-ICD nuclear access is not γ-secretase–dependent, as trans- multiple myeloma and mantle cell lymphoma (34, 57). Using lational initiation in the aberrant NOTCH2 transcripts lies C transcriptome sequencing analysis, we identified a G193R mu- terminal of the S3 cleavage site (7). We performed proof-of-concept tation within the RBPJ gene, which encodes an essential com- experiments (Fig. 5 and SI Appendix,Fig.S14) using either engi- ponent of the Notch transcription complex; this mutation confers neered human T-ALL cell lines expressing N1-ICD or the TNBC resistance to CB-103 when introduced into Notch-driven T-ALL cell line HCC1187, which has a NOTCH2 gene rearrangement. No- cells (Fig. 2). Notably, this mutant abrogated the ability of CB- tably,CB-103inhibitedgrowthofbothcelllinesinvitroaswellasthe 103 to inhibit the formation of Notch transcription complexes, as growth of HCC1187 xenotransplants, whereas GSIs were inactive. assessed by immunoprecipitation and chromatin immunopre- Previous studies also identified compounds (SAHM1, IMR-1, cipitation (ChIP) on endogenous Notch-response sites near and RIN1) that were claimed to target the Notch transcription

Lehal et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 30, 2021 A B

D C

H F

Fig. 5. CB-103 impedes growth of NOTCH+ cancer cell lines and primary human T-ALL. (A) Growth kinetics of HCC-1187 cells treated with DMSO, GSI − − (RO4929097), and CB-103 (10 μM each) for 6 d. (B) Luciferase-expressing HCC-1187 cells were subcutaneously transplanted into NOD/SCIDγc / (NSG) mice and treated with either vehicle (n = 6) or CB-103 (n = 6) for 15d (2× a day). Bioluminescence was measured 30 d posttransplantation. HCC-1187 tumor volume (mm3) measured over time in xenotransplanted mice treated with either vehicle or CB-103 (25 mg/kg) administrated twice a day (n = 6 for each group) is shown. Two independent experiments were performed. Statistical analysis was performed using two-way ANOVA, ****P < 0.00001. (C) Representative H&E staining (Left) and immunostaining for Cyclin D1 (Right) of tumors harvested from vehicle- and CB-103–treated animals is shown. (D) Oxaliplatin-resistant M43 colorectal cancer cells were transplanted into NSG mice and subsequently treated with either vehicle (n = 18), CB-103 (n = 18), oxaliplatin, or oxaliplatin and CB-103 for 12 d (25 mg/kg 1× a day) and tumor growth fold-change was monitored over time (Left) and at end stage of the experiment (Right). One-way ANOVA, Tukey’s multiple comparisons test *P < 0.05; ***P < 0.0003; ****P < 0.0001; ns, not significant. (E) Response to CB-103 of T-ALL cells in vitro. Patient- derived xenografts (n = 20) were maintained on human mesenchymal stromal cells and incubated with 0.01, 0.1, 1, 10, or 25 μM CB-103 for 7 d. Graph

represents IC50 values of each patient sample treated with CB-103. Blue circles represent patient samples positive for the presence N1-ICD, red circles represent patient samples negative for N1-ICD. (F) Immunoblots underneath show N1-ICD levels decrease upon treatment with CB-103. N1-ICD detection in indicated patient samples (numbered) on Western blot after 72-h exposure to DMSO or CB-103 is shown. Mutation status for NOTCH1 and FBXW7 are indicated. (G) Event-free survival analysis after treatment of leukemia xenografts of the T-ALL corresponding to case 1. Kaplan–Meier survival curve is shown for xenografted NSG mice. Treatment with vehicle or CB-103 as indicated when 10% of T-ALL cells were detected in peripheral blood by flow cytometry. Log-rank (Mantel–Cox) test and P value as indicated. Bar graph represents detection levels of N1-ICD in T-ALL cells after in vivo treatment with CB-103. The mean ratio (±SEM) of N1-ICD (Val-1744) determined in vehicle and CB-103–treated animals (two per condition). A representative Western blot example for N1-ICD of treated animals is shown. (H) Bar graphs show results of a second independent N1-ICD+ PDX model xenografted into NSG mice. Vehicle control and CB-103 treatment groups were subdivided into high and low tumor burden groups based on >20% and <2% of T-ALL cells detected in peripheral blood by flow cytometric analysis at treatment initiation. Animal groups were treated for 12 d with either vehicle control or CB-103. Bar graphs show absolute numbers of huCD45+CD7+ T-ALL cells after treatment. Animals with high tumor burden: n = 5 for vehicle control and n = 3 for CB-103–treated animals; animals with low tumor burden: n = 5 for both vehicle and CB-103 treated animals. Statistical analysis: Student’s t test test, *P < 0.05.

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1922606117 Lehal et al. Downloaded by guest on September 30, 2021 complex (29–31). However, in reassessing the activity of genes, such as Olmf4 and Hes1, were both down-regulated by SAHM1, we were unable to confirm any “on-Notch” inhibitory CB-103 and GSI, indicating that CB-103 reached the target tis- effects in reporter gene assays or T-ALL cells (SI Appendix, Fig. sue. In contrast, Atoh1 transcripts were significantly higher in S7). IMR-1 is a small chemical compound that was identified GSI-treated animals (Fig. 4). This distinction was confirmed in based on the strategy of inhibiting MAML1 recruitment to the intestinal organoid cultures, excluding the possibility that this Notch transcription complex (30). A side-by-side comparison of result is a consequence of variation in absorption or pharmacoki- CB-103, the GSI LY3039478, SAHM1, and IMR-1 for their netic properties in vivo. LY3039478-treated organoids displayed a ability to block growth of a Notch-driven human T-ALL cell line very different morphology than CB-103–treated cultures, showing showed that IMR-1 had no effect on Notch target gene expres- increased goblet cell differentiation and decreased proliferation sion or cell growth at doses up to 10 μM, whereas CB-103 and compared to vehicle- or CB-103–treated organoids (SI Appendix, GSI were active (SI Appendix, Fig. S7). IMR-1 evidently can Fig. S13). The mechanisms underlying this distinction are currently inhibit Notch signaling at higher concentrations, as previously unknown. Several possibilities can be considered. First, CB-103 reported (30, 59, 60). RIN1 is another small molecule that was inhibits protein–protein interaction and might therefore produce recently reported to modulate the Notch transcription complex more incomplete Notch inhibition than a potent GSI. A second (31). This small chemical compound was identified in a reporter- possibility, which is not mutually exclusive, is that CB-103 may in- based cell culture assay aimed at identifying inhibitors of inhibit only a subset of RBPJ complexes, leading to variation in the teractions between RBPJ and SHARP, which is a component responsiveness of different Notch target genes. Nevertheless, in of a transcriptional repressor complex that binds RBPJ in the Notch-driven tumors, where the levels of signaling are well above absence of NICD. In line with this possibility, RIN1-treated that of normal tissues due to activating mutations, CB-103 is evi- cells up-regulated Notch target genes, mimicking the effects of dently potent enough to produce responses in preclinical models. RBPJ knockdown. The activity of RIN1 has yet to be assessed, Further work is necessary to parse out these distinctions. in regard to therapeutic efficacy and intestinal toxicity (31), In summary, our discovery and characterization of CB-103 and comparative evaluation with CB-103 warrants future makes a compelling case that small-molecule inhibitors can be consideration. developed and used to block TF complexes, which are down- One of the major dose-limiting toxicities and hurdles to the stream of many aberrant signaling cascades, but have been his- therapeutic application of the pan-Notch inhibitor has been in- torically intransigent to therapeutic targeting. CB-103 interferes testinal toxicity (22, 23). Genetic studies in mice show that Notch with the Notch TF complex, and may thereby convey a more acts as a stem and progenitor cell gate keeper and is important for secretory versus absorptive cell fate differentiation. Condi- favorable therapeutic window than previous Notch-targeting APPLIED BIOLOGICAL SCIENCES tional, gut-specific inactivation of Notch1 and -2, the ligands Dll1 agents. Motivated by this knowledge and by its pharmacologi- and Dll4, and Rbpj result in the loss of proliferative crypt pro- cal characteristics, CB-103 is currently being evaluated in phase I/II genitors and conversion into postmitotic goblet cells (40, 41, 47). clinical trials (https://clinicaltrials.gov/ct2/show/NCT03422679). A similar phenotype has been observed in mice with simulta- Materials and Methods neous gut-specific inactivation of the Notch target genes Hes1, Hes3, and Hes5, indicating Notch signaling regulates intestinal Details of materials and methods are provided in SI Appendix, including homeostasis at least in part through Hes genes (49). Conversely, sources of constructs, compounds, cell lines and cell culture conditions (ex vivo, in vitro), luciferase reporter assay, assays of cell cycle, proliferation, cell transgenic Notch gain-of-function experiments cause a reciprocal viability, stable transformants, and animal studies. Details of microscopy, phenotype consisting of a block in secretory cell differentiation image processing and analysis, FACS and computational docking studies are and an expansion of immature crypt progenitors (61). The gut described in SI Appendix. Methods for Western blot, immunoprecipitation phenotype created by knockout of Notch pathway components is and ChIP, qRT-PCR, in situ hybridization, histology and immunostaining, RNA also observed in rodents treated with potent GSIs, such as sequencing and SLAM-seq, and bioinformatic and statistical analyses are also dibenzazepine (47), suggesting that the gut toxicity caused by provided in SI Appendix. GSI is mostly driven by Notch inhibition (16). In this context, it is interesting to note that four different γ-secretase complexes exist Ethics Statement. All animal work was carried out in accordance with Swiss and that most available GSIs block all complexes, which is likely national guidelines. This study was reviewed and approved by the cantonal to account for the intestinal toxicities in GSI-treated animals or veterinary service, Vaud. patients. A recent report showed that selective pharmacological inhibition of one (presenilin-1) of the four (PSEN) γ-secretase Data Availability Statement. All data generated in this study are included in subclasses is effective in reducing the leukemic burden of PSEN- the paper and SI Appendix. The SLAM-seq data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https:// 1 expressing T-ALL cells in xenotransplantation assays without www.ncbi.nlm.nih.gov/geo (accession no. GSE148228). causing intestinal toxicity (62). Thus, selective inhibition of the γ -secretase complex might be a potential therapeutic strategy for ACKNOWLEDGMENTS. We thank the team of the Biomolecular Screening safely targeting Notch-driven tumors, provided Notch cleavage is Facility, Ecole Polytechnique Fédérale de Lausanne, Gerardo Turcatti, Marc mostly mediated by specific PSEN subclasses. Chambon, Nathalie Ballanfat and Manuel Bueno; Viktoria Reinmüller and These effects of GSI involve alterations in the TF Atoh1 (also Jieping Zhu for compound synthesis; Daniel Hall and Rhett Kovall for tech- known as Math1), which is a master regulator of the secretory nical support and discussion of the mode of action of CB-103; and Douglas Hanahan and Maximilien Murone for critical reading, discussions, and edit- lineage (50, 63). Conditional gut-specific inactivation of Atoh1 ing of the manuscript. Molecular graphics and analyses were performed with results in loss of all secretory cells and in the context of GSI- University of California, San Francisco Chimera (NIH P41-GM103311). This mediated Notch inhibition has been shown to be essential for work was in part supported by the Swiss National Science Foundation and goblet cell fate conversion (63). the Swiss Cancer League, the National Centre of Competence in Research Chemical Biology (F.R.), the foundation “Kind und Krebs,” the “Krebsliga Zur- Unexpectedly, in vivo administration of CB-103 to mice did ich,” the Swiss National Science Foundation (310030-133108), and the clinical not lead to the anticipated goblet cell metaplasia phenotype, research focus program “Human Hemato-Lymphatic Diseases“ of the University which was observed in GSI-treated animals. Direct Notch target of Zürich. J.C.A. is supported by Harvard University Ludwig Institute.

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